Fig. 2: NAC is sufficient to increase the specificity of SRP-SR association. | Nature Communications

Fig. 2: NAC is sufficient to increase the specificity of SRP-SR association.

From: A ribosome-associated chaperone enables substrate triage in a cotranslational protein targeting complex

Fig. 2

a Scheme of the FRET assay to measure the interaction between SRP (gray with SRP54 shown in orange, NG-domains and M-domains of SRP54 are indicated) and SR (green). Green and red stars denote the donor and acceptor dyes, respectively. b Representative fluorescence time traces of SRP-SR association, measured as described in the Methods section. The fluorescence signal was normalized to the intensity at time 0 in each trace. The time traces were fit to an exponential decay function to obtain the observed rate constant, kobs, at each SR concentration. The inset shows the plot of kobs against SR concentration, which was fit to Eq. (1) to obtain kon, the association rate constant for SRP-SR binding. The range of [SR] is from 31.3 to 250 nM. c Summary of the effects of HSW on the kon values for SRP-SR binding on RNC(ss), RNC(ssmt), and RNC(ssmt2). The +HSW(RRL) and +HSW(WG) reactions contained RPBs obtained from 200 nM RRL or wheat germ ribosome, respectively. N.D., not determined. d Representative time traces of SRP-SR association on RNC(ss) and RNC(ssmt) in the absence and presence of NAC. All measurements contained 200 nM RNC, 10 nM SRP, 300 nM SR, and 300 nM NAC where indicated. Fluorescence signal was normalized to the intensity at time 0 in each measurement. e Dose-dependent effects of NAC on the apparent rate constants of SRP-SR association on RNC(ss) and RNC(ssmt). The effects of the ribosome binding mutant of NAC (NACmt) are shown in gray and black. The reactions contained 200 nM RNC, 10 nM SRP, 300 nM SR, and indicated concentrations of NAC or NACmt. f Observed association rate constants for SRP-SR binding (kobs) were plotted against SR concentration and fit to Eq. (1) to obtain values of kon. The reactions contained 200 nM RNC, 10 nM SRP, indicated concentrations of SR, and 300 nM NAC where indicated. g Summary of the effects of NAC on SRP-SR association rate constants on RNC(wt), RNC(ss), RNC(ssmt), and RNC(ssmt2). All values are shown as mean ± SD or mean with individual data points, with n = 3–5 independent measurements on the same biological sample. Source data for b–g are provided in the Source Data file.

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